Inbred corn line NPAF4467

ABSTRACT

Basically, this invention provides for an inbred corn line designated NPAF4467, methods for producing a corn plant by crossing plants of the inbred line NPAF4467 with plants of another corn plants. The invention relates to the various parts of inbred NPAF4467 including culturable cells. This invention also relates to methods for introducing transgenic transgenes into inbred corn line NPAF4467 and plants produced by said methods.

FIELD OF THE INVENTION

This invention is in the field of corn breeding, specifically relatingto an inbred corn line designated NPAF4467. This invention also is inthe field of hybrid maize production employing the present inbred.

BACKGROUND OF THE INVENTION

The original maize plant was indigenous to the Western Hemisphere. Theplants were weed like and only through the efforts of early breederswere cultivated crop species developed. The crop cultivated by earlybreeders, like the crop today, could be wind pollinated. The physicaltraits of maize are such that wind pollination results inself-pollination or cross-pollination between plants. Each maize planthas a separate male and female flower that contributes to pollination,the tassel and ear, respectively. Natural pollination occurs when windtransfers pollen from tassel to the silks on the corn ears. This type ofpollination has contributed to the wide variation of maize varietiespresent in the Western Hemisphere.

The development of a planned breeding program for maize only occurred inthe last century. A large part of the development of the maize productinto a profitable agricultural crop was due to the work done by landgrant colleges. Originally, maize was an open pollinated variety havingheterogeneous genotypes. The maize farmer selected uniform ears from theyield of these genotypes and preserved them for planting the nextseason. The result was a field of maize plants that were segregating fora variety of traits. This type of maize selection led to Summary of theNPAF4467 incremental increases in seed yield.

Large increases in seed yield were due to the work done by land grantcolleges that resulted in the development of numerous hybrid cornvarieties in planned breeding programs. Hybrids were developed frominbreds which were developed by selecting corn lines and selfing theselines for several generations to develop homozygous pure inbred lines.One selected inbred line was emasculated and another selected inbredline pollinated the emasculated inbred to produce hybrid seed F1 on theemasculated inbred line. Emasculation of the inbred usually is done bydetasseling the seed parent; however, emasculation can be done in anumber of ways. For example an inbred could have a male sterility factorwhich would eliminate the need to detassel the inbred.

In the early seventies the hybrid corn industry attempted to introduceCMS (cytoplasmic male sterility) into a number of inbred lines.Unfortunately, the CMS inbreds also introduced some very poor agronomicperformance traits into the hybrid seed which caused farmers concerncausing the maize industry to shy away from CMS material for a couple ofdecades thereafter.

However, in the last 10-15 years a number of different male sterilitysystems for maize have been successfully deployed. The mosttraditionally of these male sterility and/or CMS systems for maizeparallel the CMS type systems that have been routinely used in hybridproduction in sunflower.

In the standard CMS system there are three different maize linesrequired to make the hybrid. First, there is a cytoplasmic male-sterileline usually carrying the CMS or some other form of male sterility. Thisline will be the seed producing parent line. Second, there must be afertile inbred line that is the same or isogenic with the seed producinginbred parent but lacking the trait of male sterility. This is amaintainer line needed to make new inbred seed of the seed producingmale sterile parent. Third there is a different inbred which is fertile,has normal cytoplasm and carries a fertility restoring gene. This lineis called the restorer line in the CMS system. The CMS cytoplasm isinherited from the maternal parent (or the seed producing plant);therefore for the hybrid seed produced on such plant to be fertile thepollen used to fertilize this plant must carry the restorer gene. Thepositive aspect of this is that it allows hybrid seed to be producedwithout the need for detasseling the seed parent. However, this systemdoes require breeding of all three types of lines: 1) male sterile-tocarry the CMS: 2) the maintainer line; and, 3) the line carrying thefertility restorer gene. Use of a switchable male sterility gene reducesthe complexity of the sterility system.

In other instances, sterile hybrids are produced and the pollennecessary for the formation of grain on these hybrids is supplied byinterplanting of fertile inbreds in the field with the sterile hybrids.

Whether the seed producing plant is emasculated due to detasseling orCMS or transgenes, the seed produced by crossing two inbreds in thismanner is hybrid seed. This hybrid seed is F1 hybrid seed. The grainproduced by a plant grown from a F1 hybrid seed is referred to as F2 orgrain. Although, all F1 seed and plants, produced by this hybrid seedproduction system using the same two inbreds should be substantially thesame, all F2 grain produced from the F1 plant will be segregating maizematerial.

The hybrid seed production produces hybrid seed which is heterozygous.The heterozygosis results in hybrid plants, which are robust andvigorous plants. Inbreds on the other hand are mostly homozygous. Thishomozygosity renders the inbred lines less vigorous. Inbred seed can bedifficult to produce since the inbreeding process in corn linesdecreases the vigor. However, when two inbred lines are crossed, thehybrid plant evidences greatly increased vigor and seed yield comparedto open pollinated, segregating maize plants. An important consequenceof the homozygosity and the homogenity of the inbred maize lines is thatall hybrid seed produced from any cross of two such elite lines will bethe same hybrid seed and make the same hybrid plant. Thus the use ofinbreds makes hybrid seed which can be reproduced readily.

The ultimate objective of the commercial maize seed companies is toproduce high yielding, agronomically sound plants that perform well incertain regions or areas of the Corn Belt. To produce these types ofhybrids, the companies must develop inbreds, which carry needed traitsinto the hybrid combination. Hybrids are not often uniformly adapted forthe entire Corn Belt, but most often are specifically adapted forregions of the Corn Belt. Northern regions of the Corn Belt requireshorter season hybrids than do southern regions of the Corn Belt.Hybrids that grow well in Colorado and Nebraska soils may not flourishin richer Illinois and Iowa soils. Thus, a variety of major agronomictraits is important in hybrid combination for the various Corn Beltregions, and has an impact on hybrid performance.

Inbred line development and hybrid testing have been emphasized in thepast half-century in commercial maize production as a means to increasehybrid performance. Inbred development is usually done by pedigreeselection. Pedigree selection can be selection in an F2 populationproduced from a planned cross of two genotypes (often elite inbredlines), or selection of progeny of synthetic varieties, open pollinated,composite, or backcrossed populations. This type of selection iseffective for highly inheritable traits, but other traits, for example,yield requires replicated test crosses at a variety of stages foraccurate selection.

Maize breeders select for a variety of traits in inbreds that impacthybrid performance along with selecting for acceptable parental traits.Such traits include: yield potential in hybrid combination; dry down;maturity; grain moisture at harvest; greensnap; resistance to rootlodging; resistance to stalk lodging; grain quality; disease and insectresistance; ear and plant height. Additionally, hybrid performance willdiffer in different soil types such as low levels of organic matter,clay, sand, black, high pH, low pH; or in different environments such aswet environments, drought environments, and no tillage conditions. Thesetraits appear to be governed by a complex genetic system that makesselection and breeding of an inbred line extremely difficult. Even if aninbred in hybrid combination has excellent yield (a desiredcharacteristic), it may not be useful because it fails to haveacceptable parental traits such as seed yield, seed size, pollenproduction, good silks, plant height, etc.

To illustrate the difficulty of breeding and developing inbred lines,the following example is given. Two inbreds compared for similarity of29 traits differed significantly for 18 traits between the two lines. If18 simply inherited single gene traits were polymorphic with genefrequencies of 0.5 in the parental lines, and assuming independentsegregation (as would essentially be the case if each trait resided on adifferent chromosome arm), then the specific combination of these traitsas embodied in an inbred would only be expected to become fixed at arate of one in 262,144 possible homozygous genetic combinations.Selection of the specific inbred combination is also influenced by thespecific selection environment on many of these 18 traits which makesthe probability of obtaining this one inbred even more remote. Inaddition, most traits in the corn genome are regrettably not singledominant genes but are multi-genetic with additive gene action notdominant gene action. Thus, the general procedure of producing a nonsegregating F1 generation and self pollinating to produce a F2generation that segregates for traits and selecting progeny with thevisual traits desired does not easily lead to an useful inbred. Greatcare and breeder expertise must be used in selection of breedingmaterial to continue to increase yield and the agronomics of inbreds andresultant commercial hybrids.

Certain regions of the Corn Belt have specific difficulties that otherregions may not have. Thus the hybrids developed from the inbreds haveto have traits that overcome or at least minimize these regional growingproblems. Examples of these problems include in the eastern corn beltGray Leaf Spot, in the north cool temperatures during seedlingemergence, in the Nebraska region CLN (Corn Lethal Necrosis) and in thewest soil that has excessively high pH levels. The industry oftentargets inbreds that address these issues specifically forming nicheproducts. However, the aim of most large seed producers is to provide anumber of traits to each inbred so that the corresponding hybrid can beuseful in broader regions of the Corn Belt. The new biotechnologytechniques such as Microsatellites, RFLPs, RAPDs and the like haveprovided breeders with additional tools to accomplish these goals.

The present invention relates to an inbred corn line NPAF4467.Specifically, this invention relates to plants and seeds of this line.Additionally, this relates to a method of producing from this inbred,hybrid seed corn and hybrid plants with seeds from such hybrid seed.More particularly, this invention relates to the unique combination oftraits that combine in corn line NPAF4467.

Generally then, broadly the present invention includes an inbred cornseed designated NPAF4467. This seed produces a corn plant.

The invention also includes the tissue culture of regenerable cells ofNPAF4467 wherein the cells of the tissue culture regenerates plantscapable of expressing the genotype of NPAF4467. The tissue culture isselected from the group consisting of leaf, pollen, embryo, root, roottip, guard cell, ovule, seed, anther, silk, flower, kernel, ear, cob,husk and stalk, cell and protoplast thereof. The corn plant regeneratedfrom NPAF4467 or any part thereof is included in the present invention.The present invention includes regenerated corn plants that are capableof expressing NPAF4467's genotype, phenotype or mutants or variantsthereof.

The invention extends to hybrid seed produced by planting, inpollinating proximity which includes using preserved maize pollen asexplained in U.S. Pat. No. 5,596,838 to Greaves, seeds of corn inbredlines NPAF4467 and another inbred line if preserved pollen is not used;cultivating corn plants resulting from said planting; preventing pollenproduction by the plants of one of the inbred lines if two are employed;allowing cross pollination to occur between said inbred lines; andharvesting seeds produced on plants of the selected inbred. The hybridseed produced by hybrid combination of plants of inbred corn seeddesignated NPAF4467 and plants of another inbred line are apart of thepresent invention. This inventions scope covers hybrid plants and theplant parts including the grain and pollen grown from this hybrid seed.

The invention further includes a method of hybrid F1 production. A firstgeneration (F1) hybrid corn plant produced by the process of plantingseeds of corn inbred line NPAF4467; cultivating corn plants resultingfrom said planting; permitting pollen from another inbred line to crosspollinate inbred line NPAF4467; harvesting seeds produced on plants ofthe inbred; and growing a harvested seed are part of the method of thisinvention.

The present invention also encompasses a method of introducing at leastone targeted trait into maize inbred line comprising the steps of: (a)crossing plant grown from the present invention seed which is therecurrent parent, representative seed of which has been deposited, withthe donor plant of another maize line that comprises at least one targettrait selected from the group consisting of male sterility, herbicideresistance, insect resistance, disease resistance, amylose starch, andwaxy starch to produce F1 plants; (b) selecting from the F1 plants thathave at least one of the targeted traits, forming a pool of progenyplants with the targeted trait; (c) crossing the pool of progeny plantswith the present invention which is the recurrent parent to producebackcrossed progeny plants with the targeted trait; (d) selecting forbackcrossed progeny plants that have at least one of the target traitsand physiological and morphological characteristics of maize inbred lineof the recurrent parent, listed in Table 1 as traits of the presentinvention forming a pool of selected backcrossed progeny plants; and (e)crossing the selected backcrossed progeny plants to the recurrent parentand selecting from the resulting plants for the targeted trait andphysiological and morphological characteristics of maize inbred line ofthe recurrent parent, listed in Table 1 and reselecting from the pool ofresulting plants and repeating the crossing to the recurrent parent andselecting step in succession to form a plant that comprise the desiredtrait and all of the physiological and morphological characteristics ofmaize inbred line of the recurrent parent if the present inventionlisted in Table 1 as determined at the 5% significance level when grownin the same environmental conditions.

This method and the following method of introducing traits can be donewith less back crossing events if the trait and/or the genotype of thepresent invention are selected for or identified through the use ofmarkers. SSR, microsatellites, SNP and the like decrease the amount ofbreeding time required to locate a line with the desired trait or traitsand the characteristics of the present invention. Backcrossing in two oreven three traits (for example the glyphosate, Europe corn borer, cornrootworm resistant genes) is routinely done with the use of markerassisted breeding techniques. This introduction of transgenes ormutations into a maize line is often called single gene conversion.Although, presently more than one gene particularly transgenes ormutations which are readily tracked with markers can be moved during thesame “single gene conversion” process, resulting in a line with theaddition of more targeted traits than just the one, but still having thecharacteristics of the present invention plus those characteristicsadded by the targeted traits.

The method of introducing a desired trait into maize inbred linecomprising: (a) crossing plant grown from the present invention seed,representative seed of which has been deposited the recurrent parent,with plant of another maize line that comprises at least one targettrait selected from the group consisting of nucleic acid encoding anenzyme selected from the group consisting of phytase, stearyl-ACPdesaturase, fructosyltransferase, levansucrase, amylase, invertase,starch synthase, branching enzyme, and debranching enzyme, and starchbranching enzyme, the donor parent to produce F1 plants; (b) selectingfor the targeted trait from the F1 plants, forming a pool of progenyplants; (c) crossing the progeny plants with the recurrent parent toproduce backcrossed progeny plants; (d) selecting for backcrossedprogeny plants that have at least one of the target trait andphysiological and morphological characteristics of maize inbred line ofthe present invention as listed in Table 1 for the invention forming apool of backcrossed progeny plants; and repeating a step of crossing thenew pool with the recurrent parent and selecting for the targeted traitand the recurrent parents characteristics until the selected plant isessentially the recurrent parent with the targeted trait or targetedtraits. This selection and crossing may take at least 4 backcrosses ifmarker assisted breeding is not employed.

The inbred line and seed of the present invention are employed to carrythe agronomic package into the hybrid. Additionally, the inbred line isoften carrying transgenes that are introduced in to the hybrid seed.

Likewise included is a first generation (F1) hybrid corn plant producedby the process of planting seeds of corn inbred line NPAF4467;cultivating corn plants resulting from said planting; permitting pollenfrom inbred line NPAF4467 to cross pollinate another inbred line;harvesting seeds produced on plants of the inbred; and growing a plantfrom such a harvested seed.

A number of different techniques exist which are designed to avoiddetasseling in maize hybrid production. Some examples are switchablemale sterility, lethal genes in the pollen or anther, inducible malesterility, male sterility genes with chemical restorers. There arenumerous patented means of improving upon the hybrid production system.Some examples include U.S. Pat. No. 6,025,546, which relates to the useof tapetum-specific promoters and the barnase gene to produce malesterility; and U.S. Pat. No. 6,627,799 relates to modifying stamen cellsto provide male sterility. Therefore, one aspect of the currentinvention concerns the present invention comprising one or more gene(s)capable of restoring male fertility to male-sterile maize inbreds orhybrids and/or genes or traits to produce male sterility in maizeinbreds or hybrids.

The inbred corn line NPAF4467 and at least one transgenic gene adaptedto give NPAF4467 additional and/or altered phenotypic traits are withinthe scope of the invention. Such transgenes are usually associated withregulatory elements (promoters, enhancers, terminators and the like).Presently, transgenes provide the invention with traits such as insectresistance, herbicide resistance, disease resistance increased ordeceased starch or sugars or oils, increased or decreased life cycle orother altered trait.

The present invention includes inbred corn line NPAF4467 and at leastone transgenic gene adapted to give NPAF4467 modified starch traits.Furthermore this invention includes the inbred corn line NPAF4467 and atleast one mutant gene adapted to give modified starch, acid or oiltraits, i.e. amylase, waxy, amylose extender, starch synthase (SS) SSI,SSIIa, SSIIIa, SSIIIb, SSIVa, SSIVb, branching enzyme (BE), BEI, BEIIa,BEIIb, debranching enzyme, surgary 1, or amylose. The present inventionincludes the inbred corn line NPAF4467 and at least one transgenic gene:bacillus thuringiensis, the bar or pat gene encoding Phosphinothricinacetyl Transferase, Gdha gene, GOX, VIP, EPSP synthase gene, low phyticacid producing gene, and zein. The inbred corn line NPAF4467 and atleast one transgenic gene useful as a selectable marker or a screenablemarker is covered by the present invention.

A tissue culture of the regenerable cells of hybrid plants produced withuse of NPAF4467 genetic material is covered by this invention. A tissueculture of the regenerable cells of the corn plant produced by themethod described above is also included.

DEFINITIONS

In the description and examples, which follow, a number of terms areused. In order to provide a clear and consistent understanding of thespecifications and claims, including the scope to be given such terms,the following definitions are provided.

Early Season Trait Codes

Emergence (EMRGR): Recorded when 50% of the plots in the trial are at V1(1 leaf collar) growth stage.

1=All plants have emerged and are uniform in size

3=All plants have emerged but are not completely uniform

5=Most plants have emerged with some just beginning to break the soilsurface, noticeable lack of uniformity

7=Less than 50% of the plants have emerged, and lack of uniformity isvery noticeable

9=A few plants have emerged but most remain under the soil surface.

Seedling Growth (SVGRR): Recorded between V3 and V5 (3-5 leaf stage)giving greatest weight to seedling plant size and secondary weight touniform growth. 1=Large plant size and uniform growth

3=Acceptable plant size and uniform growth

5=Acceptable plant size and might be a little non-uniform

7=Weak looking plants and non-uniform growth

9=Small plants with poor uniformity

When taking emergence notes, hybrid ratings are relative to each otherover the trial.

Purpling (PRPLR): Emergence and/or early growth rating. Purpling is morepronounced on the under sides of leaf blades especially on midribs.

1=No plants showing purple color

3=30% plants showing purple color

5=50% plants showing purple color

7=70% plants showing purple color

9=90+% plants showing purple color

Herbicide Injury (HRBDR) List the herbicide type, which is being rated.Then rated each hybrid/variety injury as indicated below.

1=No apparent reduction in biomass or other injury symptoms

5=Moderate reduction in biomass with some signs of sensitivity

9=Severe reduction in biomass with some mortality

Mid-Season Traits Codes

Heat Units to 50% Silk (HU5SN): Recorded the day when 50% of all plantswithin a plot show 2 cm or more silk protruding from the ear. Converteddays to accumulated heat units from planting.

Heat units to 50% Pollen Shed (HUPSN): Recorded the day when 50% of allplants within a plot are shedding pollen. Converted days to accumulatedheat units from planting.

Plant Height in cm (ERHTN): After pollination, recorded average plantheight of each plot. Measured from ground to base of leaf node. Three ormore locations recorded.

Plant Ear Height in cm (PLHTN): After pollination, recorded average earheight of each plot. Measured from ground to base of ear node (shank).Three or more locations should be recorded.

Root Lodging Early % (ERTLP): Early root lodging occurs up to about twoweeks after flowering and usually involves goosenecking. Counted thenumber of root lodged plants and converted to percentage. For FieldEvaluation Test plots (FET), recorded lodged plants out of 50 plantsfrom two locations in each hybrid strip, sum, and record percentage.Foliar Disease (LFDSR): Foliar disease ratings taken one month beforeharvest through harvest. The predominant disease should be listed in thetrial information and individual hybrid ratings are recorded.1=No lesions to two lesions per leaf.3=A few scattered lesions on the leaf. About five to ten percent of theleaf surface is affected.5=A moderate number of lesions are on the leaf. About 15 to 20 percentof the leaf surface is affected.7=Abundant lesions are on the leaf. About 30 to 40 percent of the leafsurface is affected.9=Highly abundant lesions (>50 percent) on the leaf. Lesions are highlycoalesced. Plants may be prematurely killed.Data collection (as described above) on the following diseases:

Common Rust (CR) Eye Spot (ES)

Gray Leaf Spot (GLS) Northern Corn Leaf Blight (NCLB)

Stewart's Bacterial Wilt (SBW) Southern Corn Leaf Blight (SCLB)

Southern Rust (SR) Corn Virus Complex (CVC)

Preharvest Trait Codes

Heat units to Black Layer (HUBLN): Recorded the day when 50% of allplants within a plot reach black layer stage. Converted days toaccumulated heat units from planting. Taken on border rows of four-rowplots.

Harvest Population (HAVPN): Counted the number of plants in yield rows,excluding tillers, in each plot. For FET plots, count a thousandth of anacre two times and record the average.

Barren Plants (BRRNP): Counted the number of plants in yield rows havingno ears and/or abnormal ears with less than 50 kernels. For FET plots,counted barren plants out of 50 from two locations in each hybrid strip,sum, and record the percentage. Data collected on entire trial.Dropped Ears (DROPP): Counted the numbers of ears lying on the ground inyield rows. For FET plots, count dropped ears from the area of 50 plantsfrom two locations in each hybrid strip, sum, and record the percentage.Stalk Lodging % (STKLP): Stalk lodging will be reported as number ofplants broken below the ear without pushing, excluding green snappedplants. Record trials with approximately five percent or more averagestalk lodging. Counted the number of broken plants in yield rows andconverted to percent. For FET plots, counted stalk lodged plants out of50 from two locations in each hybrid strip, sum, and recorded thepercentage.Root Lodging Late % (LRTLP): Late root lodging can usually start tooccur about two weeks after flowering and involves lodging at the baseof the plant. Plants leaning at a 30-degree angle or more from thevertical are considered lodged. Counted the number of root lodged plantsin yield rows and converted to percent. For FET plots, counted rootlodged plants out of 50 from two locations in each hybrid strip, sum,and record the percentage.Push Test for Stalk and Root Quality on Erect Plants % (PSTSP): The pushtest is applied to trials with approximately five percent or lessaverage stalk lodging. Plants are pushed that are not root lodged orbroken prior to the push test. Standing next to the plant, the hand isplaced at the top ear and pushed to arm's length. Push one of the borderrows (four-row small plot) into an adjacent plot border row. Counted thenumber of plants leaning at a 30-degree angle or more from the vertical,including plants with broken stalks prior to pushing, did not countplants that have strong rinds that snap rather than bend over easily.For FET plots, push 50 plants from two interior locations of each hybridstrip, sum, and record the percentage. The goal of the push test is toidentify stalk rot and stalk lodging potential, NOT ECB injury.Data may be collected in the following manner:PUSXN: Push ten plants and enter the number of plants that do not remainupright. SPIRIT will calculate PSTSP.PSTSP: This is a percent. If you push 10 plants you can simply enter 10times the number of plants that do not remain upright (i.e. 2=20) to getthe percentage.Intactness (INTLR):1=Healthy appearance, tops unbroken5=25% of tops broken9=majority of tops brokenPlant Appearance (PLTAR): This is a visual rating based on general plantappearance taking into account all factors of intactness, pest, anddisease pressure.1=Complete plant with healthy appearance5=plants look OK9=Plants not acceptableGreen Snap (GRSNP): Counted the number of plants in yield rows thatsnapped below the ear due to brittleness associated with high winds. ForFET plots, count snapped plants out of 50 from two locations in eachhybrid strip, sum, and record the percentage.Stay-green (STGRP): This is an assessment of the ability of a grainhybrid to retain green color as maturity approaches (taken near the timeof black-layer) and should not be a reflection of hybrid maturity orleaf disease. Recorded % of green tissue.This may be listed as a Stay Green Rating instead of a Percentage.Stay Green Rating (STGRR): This is an assessment of the ability of agrain hybrid to retain green color as maturity approached (taken nearthe time of black layer or if major differences are noted later). Thisrating should not be a reflection of the hybrid maturity or leafdisease.1=solid Green Plant9=no green tissueEar/Kernel Rots (KRDSR): If ear or kernel rots are present, husk tenconsecutive ears in each plot and count the number that have evidence ofear or kernel rots, multiply by 10, and round up to the nearest ratingas described below. Identify and recorded the disease primarilyresponsible for the rot.1=No rots, 0% of the ears infected.3=Up to 10% of the ears infected.5=11 to 20% of the ears infected.7=21 to 35% of the ears infected.9=36% or more of the ears infected.Grain Quality (GRQUR): Husked back several ears after black layer stageand observed kernel cap integrity and relative amount of soft starchendosperm along the sides of kernels.1=smooth kernel caps and or 10% or less soft starch3=slight kernel wrinkles and or 30% soft starch7=moderate kernel wrinkles and or 70% soft starch9=severe kernel wrinkled and or 90% or more soft starchPreharvest Hybrid CharacteristicsEar Shape Slender, Semi-Blocky, BlockyDESHR:1=Blocky5=Semi-blocky9=SlenderDropEar Type: Fixed, Semi-Fixed, Flex (Thin outside row, every other plantfor half of row.)EARFR:1=Flex5=Semi-flex9=FixedHusk Cover: Short, Medium, LongHSKCR:1=Long5=Medium9=ShortKernel Depth: Shallow, Medium, DeepKRLNR:1=Deep5=Medium9=Short (shallow)Shank Length: Short, Medium, LongSHLNR:1=Short5=Medium9=LongCob Color (COBCR):1=White5=Pink9=Dark RedKernel Row Number: Enter average of 3 ears (KRRWN): The average numberof kernel rows on 3 ears.Cob diameter (COBDR): Cob diameter to be taken with template.

1: small

5: Medium

9: Large

Corn: Harvest Trait Codes

The following are harvest codes:

Number of Rows Harvested (NRHAN); Plot Width (RWIDN); Plot Length(RLENN); Yield Lb/Plot (GWTPN); Test Weight in Lb/Bu (TSTWN); Moisture %(MST_P); Adjusted Yield in Bu/A (YBUAN)—entered or calculated, and Yieldin Bushels per acre at standard moisture (YGSMN).

Input Form # ABR. Description Value A1 EMRGN Final number of plants perplot # A2 REGNN Region Developed: 1.Northwest 2.Northcentral #3.Northeast 4.Southeast 5.Southcentral 6.Southwest 7.Other A3 CRTYNCross type: 1.sc 2.dc 3.3w 4.msc 5.m3w 6.inbred # 7.rel. line 8.other A4KRTPN Kernel type: 1.sweet 2.dent 3.flint 4.flour 5.pop # 6.ornamental7.pipecorn 8.other A5 EMERN Days to Emergence EMERN #Days B1 ERTLP %Root lodging: (before anthesis): #% B2 GRSNP % Brittle snapping: (beforeanthesis): #% C1 TBANN Tassel branch angle of 2nd primary lateral branchdegree (at anthesis): C10 HUPSN Heat units to 50% pollen shed: (fromemergence) #HU C11 SLKCN Silk color: #/Munsell value C12 HU5SN Heatunits to 50% silk: (from emergence) #HU C13 DSAZN Days to 50% silk inadapted zone: #Days C14 HU9PN Heat units to 90% pollen shed: (fromemergence) #HU C15 HU19N Heat units from 10% to 90% pollen shed: #HU C16DA19N Days from 10% to 90% pollen shed: #Days C2 LSPUR Leaf sheathpubescence of second leaf above the # ear (at anthesis) 1-9 (1 = none):C3 ANGBN Angle between stalk and 2nd leaf above the ear degree (atanthesis): C4 CR2LN Color of 2nd leaf above the ear (at anthesis):#/Munsell value C5 GLCRN Glume Color: #/Munsell value C6 GLCBN Glumecolor bars perpendicular to their veins # (glume bands): 1.absent2.present C7 ANTCN Anther color: #/Munsell value C8 PLQUR Pollen Shed:1-9 (0 = male sterile) # C9 HU1PN Heat units to 10% pollen shed: (fromemergence) #HU D1 LAERN Number of leaves above the top ear node: # D10LTBRN Number of lateral tassel branches that originate # from thecentral spike: D11 EARPN Number of ears per stalk: # D12 APBRRAnthocyanin pigment of brace roots: 1.absent # 2.faint 3.moderate 4.darkD13 TILLN Number of tillers: # D14 HSKCN Husk color 25 days after 50%silk: (fresh) #/Munsell value D2 MLWVR Leaf marginal waves: 1-9 (1 =none) # D3 LFLCR Leaf longitudinal creases: 1-9 (1 = none) # D4 ERLLNLength of ear leaf at the top ear node: #cm D5 ERLWN Width of ear leafat the top ear node at the #cm widest point: D6 PLHTN Plant height totassel tip: #cm D7 ERHCN Plant height to the top ear node: #cm D8 LTEINLength of the internode between the ear node #cm and the node above: D9LTASN Length of the tassel from top leaf collar to tassel #cm tip: E1HSKDN Husk color 65 days after 50% silk: (dry) #/Munsell value E10 DSGMNDays from 50% silk to 25% grain moisture in #Days adapted zone: E11SHLNN Shank length: #cm E12 ERLNN Ear length: #cm E13 ERDIN Diameter ofthe ear at the midpoint: #mm E14 EWGTN Weight of a husked ear: #gm E15KRRWR Kernel rows: 1.indistinct 2.distinct # E16 KRNAR Kernel rowalignment: 1.straight 2.slightly curved # 3.curved E17 ETAPR Ear taper:1.slight 2.average 3.extreme # E18 KRRWN Number of kernel rows: # E19COBCN Cob color: #/Munsell value E2 HSKTR Husk tightness 65 days after50% silk: 1-9 # (1 = loose) E20 COBDN Diameter of the cob at themidpoint: #mm E21 YBUAN Yield: #kg/ha E22 KRTEN Endosperm type: 1.sweet2.extra sweet 3.normal 3 4.high amylose 5.waxy 6.high protein 7.highlysine 8.super sweet 9.high oil 10.other E23 KRCLN Hard endosperm color:#/Munsell value E24 ALECN Aleurone color: #/Munsell value E25 ALCPRAleurone color pattern: 1.homozygous # 2.segregating E26 KRLNN Kernellength: #mm E27 KRWDN Kernel width: #mm E28 KRDPN Kernel thickness: #mmE29 K1KHN 100 kernel weight: #gm E3 HSKCR Husk extension: 1.short (earexposed) 2.medium # (8 cm) 3.long (8-10 cm) 4.very long (>10 cm) E30KRPRN % round kernels on 13/64 slotted screen: #% E4 HEPSR Position ofear 65 days after 50% silk: 1.upright # 2.horizontal 3.pendent E5 STGRPStaygreen 65 days after anthesis: 1-9 (1 = worst) # E6 DPOPP % droppedears 65 days after anthesis: % E7 LRTRP % root lodging 65 days afteranthesis: % E8 HU25N Heat units to 25% grain moisture: (from #HUemergence) E9 HUSGN Heat units from 50% silk to 25% grain moisture in#HU adapted zone: Color Choices: 1.light green 2.medium green 3.darkgreen 4.very dark green 5.green-yellow 6.pale yellow 7.yellow8.yelow-orange 9.salmon 10.pink-orange 11.pink 12.light red 13.cherryred 14.red 15.red and white 16.pale purple 17.purple 18.colorless19.white 20.white capped 21.buff 22.tan 23.brown 24.bronze 25.variegated(describe) 26.other (describe)

DETAILED DESCRIPTION OF THE INVENTION

The inbred provides uniformity and stability within the limits ofenvironmental influence for traits as described in the VarietyDescription Information for the invention listed in Table 1 thatfollows.

The inbred has been produced through a dihaploid system or isself-pollinated for a sufficient number of generations to give inbreduniformity. During plant selection in each generation, the uniformity ofplant type was selected to ensure homozygosity and phenotypic stability.The line has been increased in isolated farmland environments with dataon uniformity and agronomic traits being observed to assure uniformityand stability. No variant traits have been observed or are expected inG07-NPAF4467.

The best method of producing the invention is by planting the seed ofG07-NPAF4467 which is substantially homozygous and self-pollinating orsib pollinating the resultant plant in an isolated environment, andharvesting the resultant seed.

TABLE 1 G07-NPAF4467 VARIETY DESCRIPTION INFORMATION #1 Type: Dent #2Region Best Adapted: Broadly adapted MG Maturity Hybrid RM Group Range(estimate) 4 98-102 102 #3 Plant Traits SyngentaCode Type AntherColorGlumeColor SilkColor NPH8431 Dent Yellow Green Red/purple stripedNPAF4467 Dent Yellow Red/purple Red/purple NP2017 Dent Green GreenRed/purple Endosperm BraceRootColor CobColor KernelColor Type ModerateRed Yellow Normal Moderate Red Yellow Red Yellow Inbred diseaseresistant levels Line GW EYE NLB SLB GLS NPAF4467 3 4 5 6 5

The data provided above is often a color. The Munsell code is areference book of color, which is known and used in the industry and bypersons with ordinary skill in the art of plant breeding. The purity andhomozygosity of inbred G07-NPAF4467 is constantly being tracked usingisozyme genotypes.

Isozyme Genotypes for G07-NPAF4467

Isozyme data were generated for inbred corn line G07-NPAF4467 accordingto procedures known and published in the art. The data in Table 2 givesthe electrophoresis data on G07-NPAF4467.

TABLE 2 ELECTROPHORESIS RESULTS FOR G07-NPAF4467 Line Code IDH2 PGD2ACP4 MDH4 MDH2 MDH5 MDH1 MDH3 NPH8431 4 2.8 4 12 3 12 6 16 NPAF4467 42.8 4 12 3 12 6 16 Line Code PGM1 MDH6 PGM2 PGD1 PHI1 IDH1 AHD1 PUR %ACP1 NPH8431 9 Mm 3 3.8 4 4 4 100 2 NPAF4467 9 Mm 3 3.8 4 4 4 100 2

Table 3A shows a comparison between G07-NPAF4467 and a comparableinbred. G07-NPAF4467 has similar yield to NPAF4449. However, the presentinvention does require significantly less heat units to reach 50%silking in the plants than the comparison inbred. The two inbreds showsignificant differences across S50 heat unit measurement with thepresent invention reaching 50% silking significantly earlier.

TABLE 3A PAIRED INBRED COMPARISON DATA Heat Units to Heat Units to YearInbred Yield Stand P50 S50 Overall NPAF4467 97.90 33285.70 1385.201382.90 NPAF4449 94.40 33000.00 1382.20 1415.10 Diff 3.50 285.70 3.0032.20 #Expts 7.00 7.00 15.00 15.00 Prob 0.71 0.73 0.72 0.097* *.05 <Prob <= .10 ** .01 < Prob <= .05 *** .00 < Prob <= .01

Table 3B shows a comparison between G07-NPAF4467 and a comparableinbred. G07-NPAF4467 has similar yield to NPAF4466. However, the presentinvention does require significantly less heat units to reach both 50%pollination and 50% silking in the plants than the comparison inbred.The present invention flowers to the 50% pollen and silk level fasterthan the comparison inbred.

TABLE 3B PAIRED INBRED COMPARISON DATA Heat Units to Heat Units to YearInbred Yield Stand P50 S50 Overall NPAF4467 97.90 33285.70 1388.301382.40 NPAF4466 85.30 32000.00 1411.40 1424.00 Diff 12.60 1285.70 23.1041.50 #Expts 7.00 7.00 21.00 21.00 Prob 0.43 0.39 0.024** 0.000*** * .05< Prob <= .10 **.01 < Prob <= .05 ***.00 < Prob <= .01

Table 4 shows the GCA (General Combining Ability) estimates ofG07-NPAF4467 compared with the GCA estimates of the other inbreds. Theestimates show the general combining ability weighted by the number ofexperiment/location combinations in which the specific hybridcombination occurs. The interpretation of the data for all traits isthat a positive comparison is a practical advantage. A negativecomparison is a practical disadvantage. The general combining ability ofan inbred is clearly evidenced by the results of the general combiningability estimates. This data compares the inbred parent in a number ofhybrid combinations to a group of “checks”. The check data is from ourcompany's and other companies' hybrids which are commercial products andpre-commercial hybrids, which were grown in the same sets and locations.

TABLE 4 % Late % Early % Stalk % Push Root Root % Dropped Parent1Parent2 N06 N07 N Yield Moisture TestWeight Lodging Test Lodging LodgingEars NPAF4467 26 26 −1.6 0.2 0.2 1.0 8.7 1.4 0.1 NPAF4467 27 27 −4.7 1.40.5 −7.6 −13.0 −1.1 −0.5 NPAF4467 26 26 −4.0 −0.3 −0.5 1.6 −1.3 4.0 0.1NPAF4467 27 27 6.9 −0.4 −0.6 2.6 8.7 3.4 −1.0 NPAF4467 27 27 7.0 1.1 0.4−5.3 −14.7 0.9 0.1 NPAF4467 25 25 1.5 0.2 0.1 −5.0 −1.3 3.7 0.1 NPAF446727 27 1.8 0.2 0.2 −6.8 −14.7 2.8 0.1 NPAF4467 38 38 7.8 1.8 0.6 −0.8 4.43.9 −6.4 0.0 XR= 38 253 2.2 0.6 0.1 −2.5 −3.6 2.4 −6.4 −0.1 XH= 38 8 1.80.5 0.1 −2.5 −2.9 2.4 −6.4 −0.1 Final Vigor Heatunits Heatunits EarPlant Parent1 Parent2 N06 N07 N Stand Emergence Rating Rating to S50 toP50 Height Height NPAF4467 26 26 −0.6 0.5 −1.0 25.3 33.7 4.0 1.8NPAF4467 27 27 −1.5 1.0 −1.3 22.3 8.4 12.8 5.8 NPAF4467 26 26 −3.1 0.7−0.5 27.9 35.9 10.2 5.4 NPAF4467 27 27 −1.2 1.1 −0.5 20.9 28.6 −2.2 1.2NPAF4467 27 27 0.1 0.0 −1.1 0.9 2.7 −2.4 11.4 NPAF4467 25 25 −0.5 0.0−0.8 43.6 52.1 6.6 10.6 NPAF4467 27 27 −1.0 0.4 −1.1 46.3 59.4 1.4 14.6NPAF4467 38 38 0.1 −0.4 0.5 −44.8 −51.3 7.1 −4.4 XR= 38 253 −0.9 0.4−0.6 19.1 22.7 4.8 5.3 XH= 38 8 −1.0 0.4 −0.7 17.8 21.2 4.7 5.8 XR = GCAEstimate: Weighted by Expt XH = GCA Estimate: Weighted by Parent2 XT =Same as XH but using only those parent2 with two years of data

Table 5 shows the inbred G07-NPAF4467 in hybrid combination, as Hybrid1, in comparison with another hybrid, which is adapted for the sameregion of the Corn Belt. When in this hybrid combination, the presentinbred G07-NPAF4467 carries significantly more yield in comparison tothe other commercial hybrid. The percent stalk lodging for thecommercial hybrid and the present invention is significantly different.

TABLE 5 PAIRED HYBRID COMPARISON DATA Hybrid Yield Moist TWT % SL % PushHybrid 1 54.3 16.6 174.4 29187.0 2.4 HYBRID 2 54.5 17.6 171.5 28724.03.1 #Expts 212.0 263.0 263.0 36.0 8.0 Diff 0.2 1.0 2.9 463.3 0.8 Prob0.2 0.000*** 0.054* 0.3 0.048** Hybrid % EarlyRL % DE FS % Greensnap %Barren Hybrid 1 0.0 7.1 4.5 0.1 4.5 HYBRID 2 0.0 3.8 4.2 0.0 6.0 #Expts21.0 62.0 76.0 55.0 2.0 Diff 0.0 3.3 0.3 0.1 1.5 Prob 0.005*** 0.5 0.20.2

Table 6 shows the yield response of G07-NPAF4467 in hybrid combinationdesignated as Hybrid 1 in comparison with the plants in the environmentaround it at the same location and in comparison with two other hybridsdesignated Hybrid 2 and Hybrid 3.

The data for the present inbred is showing results that are within theerror of the environment for the plots. However, in plot trials thepresent invention appears to trend towards higher potential yields inhigher yielding environments. However, in strip trials the presentinvention again is within the standard of error but seems to trendtoward similar yield result as the environment level.

TABLE 6 YIELD RESPONSE Environment Yield Hybrid Error 75 100 125 150 175200 Research Plots # Plots Hybrid 1 15.7 38 60 89 118 147 176 205 HYBRID2 24.3 65 43 79 114 150 185 221 Hybrid 1 15.7 38 60 89 118 147 176 205HYBRID 3 15.5 66 62 88 115 142 169 196 Strip Tests # Strips Hybrid 118.6 194 85 108 131 154 177 200 HYBRID 2 18.8 622 73 99 125 151 177 203Hybrid 1 18.6 194 85 108 131 154 177 200 HYBRID 3 17.6 130 74 99 123 148172 197

This invention also is directed to methods for producing a corn plant bycrossing a first parent corn plant with a second parent corn plantwherein the first or second parent corn plant is an inbred corn plantfrom the line NPAF4467. Further, both first and second parent cornplants can come from the inbred corn line NPAF4467 which produces a selfof the inbred invention. The present invention can be employed in avariety of breeding methods which can be selected depending on the modeof reproduction, the trait, and the condition of the germplasm. Thus,any breeding methods using the inbred corn line NPAF4467 are part ofthis invention: selfing, backcrosses, hybrid production, and crosses topopulations, and haploid by such old and known methods of using KWSinducers lines, Krasnador inducers, stock six material that induceshaploids for dihaploid formation, and anther culturing and the like.

The present invention may be useful as a male-sterile plant. Sterilitycan be produced by pulling or cutting tassels from the plant,detasseling, use of gametocides, use of genetic material to render theplant sterile using a CMS type of genetic control or a nuclear geneticsterility. Male sterility is employed in a hybrid production byeliminating the pollen from the seed producing parent so when inisolation from other pollen source the only available pollen is thatfrom the second male fertile inbred planted most often in rows near themale sterile inbred.

A number of different inventions exist which are designed to avoiddetasseling in maize hybrid production. Some examples are switchablemale sterility, lethal genes in the pollen or anther, inducible malesterility, male sterility genes with chemical restorers, sterility geneslinked with a parent. U.S. Pat. No. 6,025,546, relates to the use oftapetum-specific promoters and the barnase gene. U.S. Pat. No. 6,627,799relates to modifying stamen cells to provide male sterility. Therefore,one aspect of the current invention concerns the present inventioncomprising one or more gene(s) capable of restoring male fertility tomale-sterile maize inbreds or hybrids.

Methods for genetic male sterility are disclosed in EPO 89/3010153.8, WO90/08828, U.S. Pat. Nos. 4,654,465, 4,727,219, 3,861,709, 5,432,068 and3,710,511. Gametocides which are chemicals or substances that negativelyaffect the pollen or at least the fertility of the pollen can beemployed to provide male sterility.

Unfortunately for hybrid production nature complicates male sterilityand as a result there are self pollinated female inbred seeds in somehybrid production. Great measures are taken to avoid this inbredproduction in a hybrid field but it unfortunately does occur. If ahybrid bag of seed is tested with molecular markers it may be possibleto detect inbred seed. If the hybrid seed is planted these inbred plantstend to be readily identifiable by this runt like appearance—shorterplant, small ear. Self pollination of plants grown from these femaleinbreds seed produces the female inbred seed. This seed is not intendedto be sold to the growers for breeding but only for use as grain andforage.

The process for producing inbred seed from a hybrid seed bag comprisesplanting a group of seed comprising seed from a hybrid production, oneof the hybrid parents is the present invention, growing plants from thisseed, identifying any inbred plants, selecting and pollinating theinbred plant.

A number of well known methods can be employed to identify the genotypeof maize. The ability to understand the genotype of the presentinvention increases as the technology moves toward better markers foridentifying different components within maize genetic material. One ofthe oldest methods is the use of isozymes which provides a generalizedfootprint of the material. Other markers that were adapted to provide ahigher definition profile include Restriction Fragment LengthPolymorphisms (RFLPs), Amplified Fragment Length Polymorphisms (AFLPs),Random Amplified Polymorphic DNAs (RAPDs), Polymerase Chain Reaction(there are different types of primers or probes) (PCR), Microsattelites(SSRs), and Single Nucleotide Polymorphisms (SNPs) just to list a few.The use of these and a number of other markers for gathering genotypeinformation is well understood in the industry and can be found incollege textbooks such as Breeding Field Crops, Milton et. al. IowaState University Press.

The profile of the inbred of this invention should be close tohomozygous for alleles. A marker profile produced with any of the locusidentifying systems known in the industry will identify a particularallele at particular loci. A F1 hybrid made from the inbred of thisinvention will comprise a marker profile of the sum of both of itsinbred parents. At each locus the allele for the present invention andthe allele for the other inbred parent should be present. Thus theprofile of the present invention will permit identification of hybridsas containing the inbred parent of the present invention. To identifythe female portion of the hybrid the material from the pericarp which ismaternally inherited is employed. The comparison of this maternalprofile with the hybrid profile will allow identification of thepaternal profile. The present invention includes a maize cell that ispart of an inbred or hybrid plant which includes its seed or plant partthat has the marker profile of alleles of the present invention.

Marker systems are not just useful for identification of the presentinvention; they are also useful for breeding and trait conversiontechniques. Polymorphisms in maize permit the use of markers for linkageanalysis. If SSR are employed with flanking primers PCR can be used andSouthern Blots can often be eliminated. Use of flanking markers and PCRand amplification of the material is well known by the industry. Primersfor SSRS and mapping information are publicly available through the helpof the USDA at Maize GDB on the web.

Marker profiles of this invention can identify essentially derivedvarieties or progeny developed with the inbred in its ancestry. Thisinbred may have progeny identified by having a molecular marker profileof at least 25%, to 40%, 45%, 50% to 80% (which includes each of thenumber between these two percentages), 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% geneticcontribution of the present inbred invention, as measured by eitherpercent identity or percent similarity.

The present invention may have a new locus or trait introgressed throughdirect transformation or backcrossing or marker assisted breeding. Abackcross conversion or locus conversion both refer to a product of abackcrossing program.

DNA sequences are introduced through backcrossing (Hallauer et al. inCorn and Corn Improvement, Sprague and Dudley, Third Ed. 1998), with theinvention utilized as the recurrent parent.

When the present inbred is used as a recurrent parent in a breedingprogram it is often referred to as backcrossing. Backcrossing is oftenemployed to introgression a desired trait or trait(s), either transgenicor nontransgenic, into the recurrent parent. A plant may be selectedwith the trait or the desired locus in one or more backcrosses. Ifmarkers are employed to assist in selection the number of backcrossesneeded to recover the recurrent parent with the desired trait or locuscan be relatively few two or three. However, 3, 4, 5 or more backcrossesare often required to produce the desired inbred with the gene or lociconversion in place. The number of backcrosses needed for a traitintrogression is often linked to the genetics of the trait. Multigenictraits, recessive alleles, unlinked traits, and how the traits areinherited all play a role in the number of backcrosses that may benecessary to achieve the desired backcross conversion of the inbred.

Dominant, single gene traits or traits with obvious phenotypic changesare particularly well managed in a backcrossing program. Prior totransformation and prior to markers backcrossing was employed since atleast the 1950's to alter grain color, to move mutations intoinbreds—such as sugary 2, waxy, amylose extender, dull, brittle,shrunken, sugary 1, waxy (wx), shrunken-2,

In a book written by Hallauer entitled Corn and Corn Improvement,Sprague and Dudley, 3rd Ed. 1998 the basics of this type of crossingalong with a number of other corn breeding methods such as recurrent orbulk or mass selection, pedigree breeding, open pollination breeding,marker assisted selection, double haploids development and breeding aretaught. The ordinary corn breeder understands these breeding systems andhow to apply them to the present invention therefore repetition of thesebreeding methods need not be listed within this application.

The backcrossing program is more complicated when the trait is arecessive gene. To determine the presence of the recessive gene oftenrequires the use of additionally testing to determine if the trait hasbeen transferred. Use of markers to detect the gene reduces thecomplexity of trait identification in the progeny. A marker that is aSNP specific for the trait itself can be very useful in increasing theefficiency and speed of tracking a recessive trait within a backcrossingprogram. Backcrossing of recessive traits has been preformed since atleast the 1950 as mutant traits are often recessive. These mutant traitsare frequently moved into more elite germplasm. Mutations can resultfrom acts of nature or plant or seed or pollen exposure to temperaturealterations, culturing, radiation in various forms, chemical mutagenslike EMS and others. Some of the mutant genes which have been identifiedin maize include the genotypes: waxy (wx), amylose extender (ae), dull(du), horny (h), shrunken (sh), brittle (bt), floury (fl), opaque (O),and sugary (su). Nomenclature for mutant genes is based on the effectthese mutant genes have on the physical appearance, phenotype, of thekernel. It is also known that within these genotypes there are geneswhich produce starch with markedly different functional properties eventhough the phenotypes are the same. Such subspecies have generally beengiven a number after the named genotype, for example, sugary-1),sugary-2 (su2); shrunken 1 and shrunken 2. Traits such as Ht, waxy,shrunken, amylose extender, opaque, sugary 1, 2, dull, IT (imidazolmonetolerance), IR, sterility, fertility, phytic acid, NLB (northern leafblight), SLB (southern leaf blight), and the like have all beenintrogressed into elite inbreds through backcrossing programs. The lastbackcross generation is usually selfed if necessary to recover theinbred of interest with the introgressed trait.

All plants and plant cells produced using inbred corn line NPAF4467 arewithin the scope of this invention. The invention encompasses the inbredcorn line used in crosses with other, different, corn inbreds to produce(F1) corn hybrid seeds and hybrid plants and the grain produced on thehybrid plant. This invention includes plant and plant cells, which upongrowth and differentiation produce corn plants having the physiologicaland morphological characteristics of the inbred line NPAF4467.

Additionally, this maize can, within the scope of the invention,contain: transgenic genes such as but not limited to insect resistantgenes such as European Corn Borer resistant gene(s), Corn Rootwormgene(s), an example is event DAS-59122-7, Mir603 Modified Cry3A event,MON 89034, MON 88017 Bacillus thuringiensis (Cry genes) Cry34/35Ab1,Cry1A.105, PO Cry1F, Cry2Ab2, Cry1A, Cry1AB, Cry1Ac, Cry3Bb1, or otherBt genes with resistance to other insects. Herbicide resistant genessuch as Pat gene or Bar gene, EPSP, the altered protoporphyrinogenoxidase (protox enzyme) U.S. Pat. Nos. 5,767,373, 6,282,837, WO 01/12825and the like can be introduced into the present invention. And diseaseresistant genes such as the Mosaic virus resistant gene, etc., or traitaltering genes such as lignin genes, flowering genes, oil modifyinggenes, senescence genes and the like can be introduced in combinationwith other genes to form stacks or individual. Altered carbohydrates oraltered starch can include genes for enzymes that affects the synthase,branching enzymes, pullanases, debranching enzymes, isoamylases, alphaamylases, beta amylases, AGP, ADP and other enzymes which effect theamylose, amylopectin ratio or content or the branching pattern of starchare within the scope of this invention. The fatty acid modifying genescan also affect starch content and be introduced into the presentinvention.

The methods and techniques for inserting, or producing and/oridentifying a mutation or making or reshuffling a transgene andintrogressing the trait or gene into the present invention throughbreeding, transformation, mutating and the like are well known andunderstood by those of ordinary skill in the art.

Various techniques for breeding, moving or altering genetic materialwithin or into the present invention (whether it is an inbred or inhybrid combination) are also known to those skilled in the art. Thesetechniques to list only a few are anther culturing, haploid/doublehaploid production, (stock six, which is a breeding/selection methodusing color markers and is a haploid method that has been in use forforty years and is well known to those with skill in the art),transformation, irradiation to produce mutations, chemical or biologicalmutation agents and a host of other methods are within the scope of theinvention. All parts of the NPAF4467 plant including its plant cells(with maternal and paternal components) produced using the inbred cornline is within the scope of this invention. The term transgenic plantrefers to plants having genetic sequences, which are introduced into thegenome of a plant by a transformation method and the progeny thereof.The transgene once introduced into plant material and integrated stablycan be moved into other germplasm by standard breeding practices.

The basic steps of introducing genes into monocots have been known inthe art for years. The most common method of maize transformation isreferred to as gunning or microprojectile bombardment. The processemploys small gold-coated particles coated with DNA which are shot intothe transformable material. Detailed techniques for gunning DNA intocells, tissue, callus, embryos, and the like are well known in the priorart. One example of steps that can be involved in monocot transformationare concisely outlined in U.S. Pat. No. 5,484,956 “Fertile TransgenicZea mays Plants Comprising Heterologous DNA Encoding BacillusThuringiensis Endotoxin” issued Jan. 16, 1996 and also in U.S. Pat. No.5,489,520 “Process of Producing Fertile Zea mays Plants and ProgenyComprising a Gene Encoding Phosphinothricin Acetyl Transferase” issuedFeb. 6, 1996.

Plant cells such as maize can be transformed not only by the use of agunning device but also by a number of different techniques. Therecombinant DNA molecules of the invention can be introduced into theplant cell in a number of art-recognized ways. Those skilled in the artwill appreciate that the choice of method might depend on the type ofplant material being targeted for transformation. Suitable methods oftransforming plant cells include microinjection (Crossway et al.,BioTechniques 4:320-334 (1986)), electroporation (Riggs et al, Proc.Natl. Acad. Sci. USA 83:5602-5606 (1986), Agrobacterium mediatedtransformation (Hinchee et al., Biotechnology 6:915-921 (1988)), directgene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)), ballisticparticle acceleration using devices available from Agracetus, Inc.,Madison, Wis. and Dupont, Inc., Wilmington, Del. (see, for example,Sanford et al., U.S. Pat. No. 4,945,050; and McCabe et al.,Biotechnology 6:923-926 (1988)), protoplast transformation/regenerationmethods (see U.S. Pat. No. 5,350,689 issued Sep. 27, 1994 to Ciba-GeigyCorp.), Whiskers technology (See U.S. Pat. Nos. 5,464,765 and 5,302,523)and pollen transformation (see U.S. Pat. No. 5,629,183). Also see,Weissinger et al., Annual Rev. Genet. 22:421-477 (1988); Sanford et al.,Particulate Science and Technology 5:27-37 (1987) (onion); Christou etal., Plant Physiol. 87:671-674 (1988) (soybean); McCabe et al.,Bio/Technology 6:923-926 (1988) (soybean); Datta et al., Bio/Technology8:736-740 (1990) (rice); Klein et al., Proc. Natl. Acad. Sci. USA,85:4305-4309 (1988) (maize); Klein et al., Bio/Technology 6:559-563(1988) (maize); Klein et al., Plant Physiol. 91:440-444 (1988) (maize);Fromm et al., Bio/Technology 8:833-839 (1990); Gordon-Kamm et al., PlantCell 2:603-618 (1990) (maize); and U.S. Pat. Nos. 5,591,616 and5,679,558 (rice), for more transformation information.

A further subject of the present invention is the plants regeneratedfrom transformed cells. Regeneration is effected by any suitableprocess, which depends on the nature of the species are listed in thesepatents and patent applications which are cited in particular for theprocesses for transforming plant cells and regenerating plants are thefollowing: U.S. Pat. Nos. 4,459,355, 4,536,475, 5,464,763, 5,177,010,5,187,073, EP 267,159, EP 604 662, EP 672 752, U.S. Pat. Nos. 4,945,050,5,036,006, 5,100,792, 5,371,014, 5,478,744, 5,179,022, 5,565,346,5,484,956, 5,508,468, 5,538,877, 5,554,798, 5,489,520, 5,510,318,5,204,253, 5,405,765, EP 442 174, EP 486 233, EP 486 234, EP 539 563, EP674 725, WO 91/02071 and WO 95/06128.

The use of pollen, cotyledons, zygotic embryos, meristems and ovum asthe target issue can eliminate the need for extensive tissue culturework. Generally, cells derived from meristematic tissue are useful. Themethod of transformation of meristematic cells of cereal is taught inthe PCT application WO96/04392. Any number of various cell lines,tissues, calli and plant parts can and have been transformed by thosehaving knowledge in the art. Methods of preparing meristems, callus orprotoplasts from various plants are well known in the art and specificmethods are detailed in patents and references used by those skilled inthe art. The requirement of the plant material is that it can ultimatelybe used to form a transformed plant.

The DNA used for transformation of plants may be circular, linear, anddouble or single stranded. Usually, the DNA is in the form of a plasmid.The plasmid usually contains regulatory and/or targeting sequences whichassists the expression or targeting of the gene in the plant. Themethods of forming plasmids for transformation are known in the art.Plasmid components can include such items as: leader sequences, transitpolypeptides, promoters, terminators, genes, introns, marker genes, etc.The structures of the gene orientations can be sense, antisense, partialantisense, or partial sense: multiple gene copies or artificial genechromosomes can be used. The transgene can come from various non-plantgenes (such as; bacteria, yeast, animals, and viruses) along with beingfrom plants.

The regulatory promoters employed can be constitutive such as CaMv35Spromoter, rice action promoter, and polyubiquitin promoter U.S. Pat. No.5,614,399, for monocots or tissue specific promoters such as CABpromoters, MR7 described in U.S. Pat. No. 5,837,848, etc. The prior artpromoters, includes but is not limited to, octopine synthase, nopalinesynthase, CaMv19S, mannopine synthase, Figwort mosaic virus promoter,etc. These regulatory sequences can be combined with introns,terminators, enhancers, leader sequences and the like in the materialused for transformation.

The isolated DNA is then transformed into the plant. A gene introgressedinto this invention typically comprises a nucleotide sequence whoseexpression is responsible or contributes to the trait under the controlof a promoter appropriate for the expression of the nucleotide sequenceat the desired time in the desired tissue or part of the plant.Constitutive or inducible promoters are used. The sequence may alsocomprise other regulatory elements such as for example translationenhancers or termination signals. In an embodiment, the nucleotidesequence is the coding sequence of a gene and is transcribed andtranslated into a protein. In another embodiment, the nucleotidesequence encodes an antisense RNA, a sense RNA that is not translated oronly partially translated, a t-RNA, a r-RNA or a sn-RNA.

The genes responsible for a specific gene trait are generally inheritedthrough the nucleus. Known exceptions are, e.g. the genes for malesterility, some of which are inherited cytoplasmically, but still act assingle gene traits. In an embodiment, a heterologous transgene isintegrated into the nuclear genome of the donor, non-recurrent parent.In another embodiment, a heterologous transgene to be transferred tointo present invention is integrated into the plastid genome of a donor,non-recurrent parent. In an embodiment, a plastid transgene comprisesone gene transcribed from a single promoter or two or more genestranscribed from a single promoter.

In an other embodiment, a transgene whose expression results orcontributes to a desired trait to be transferred to the presentinvention comprises a virus resistance trait such as, for example, aMDMV strain B coat protein gene whose expression confers resistance tomixed infections of maize dwarf mosaic virus and maize chlorotic mottlevirus in transgenic maize plants (Murry et al. Biotechnology (1993)11:1559 64). In another embodiment, a transgene comprises a geneencoding an insecticidal protein, such as, for example, a crystalprotein of Bacillus thuringiensis or a vegetative insecticidal proteinfrom Bacillus cereus, such as VIP3 (see for example Estruch et al. NatBiotechnol (1997) 15:137 41). Also see, U.S. Pat. Nos. 5,877,012,6,291,156; 6,107,279 6,291,156 and 6,429,360. In another embodiment, aninsecticidal gene introduced into the present invention is a Cry1Ab geneor a portion thereof, for example introgressed into present inventionfrom a maize line comprising a Bt-11 event as described in U.S. Pat. No.6,114,608, which is incorporated herein by reference, or from a maizeline comprising a 176 event as described in Koziel et al. (1993)Biotechnology 11: 194 200. In yet another embodiment, a transgeneintrogressed into the present invention comprises a herbicide tolerancegene. For example, expression of an altered acetohydroxyacid synthase(AHAS) enzyme confers upon plants tolerance to various imidazolinone orsulfonamide herbicides (U.S. Pat. No. 4,761,373). In another embodiment,a non-transgenic trait conferring tolerance to imidazolinones isintrogressed into present invention (e.g. a “IT” or “IR” trait). U.S.Pat. No. 4,975,374, relates to plant cells and plants containing a geneencoding a mutant glutamine synthetase (GS) resistant to inhibition byherbicides that are known to inhibit GS, e.g. phosphinothricin andmethionine sulfoximine. Also, expression of a Streptomyces bar geneencoding a phosphinothricin acetyl transferase in maize plants resultsin tolerance to the herbicide phosphinothricin or glufosinate (U.S. Pat.No. 5,489,520). U.S. Pat. No. 5,013,659, which is incorporated herein byreference, is directed to plants that express a mutant acetolactatesynthase (ALS) that renders the plants resistant to inhibition bysulfonylurea herbicides. U.S. Pat. No. 5,162,602 discloses plantstolerant to inhibition by cyclohexanedione and aryloxyphenoxypropanoicacid herbicides. The tolerance is conferred by an altered acetylcoenzyme A carboxylase (ACCase). U.S. Pat. No. 5,554,798 disclosestransgenic glyphosate tolerant maize plants, which tolerance isconferred by an altered 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthasegene. U.S. Pat. No. 5,804,425 discloses transgenic glyphosate tolerantmaize plants, which tolerance is conferred by an EPSP synthase genederived from Agrobacterium tumefaciens CP-4 strain. Also, tolerance to aprotoporphyrinogen oxidase inhibitor is achieved by expression of atolerant protoporphyrinogen oxidase enzyme in plants (U.S. Pat. No.5,767,373). Another trait transferable to the present invention confersa safening effect or additional tolerance to an inhibitor of the enzymehydroxyphenylpyruvate dioxygenase (HPPD) and transgenes conferring suchtrait are, for example, described in WO 9638567, WO 9802562, WO 9923886,WO 9925842, WO 9749816, WO 9804685 and WO 9904021. All issued patentsreferred to herein are, in their entirety, expressly incorporated hereinby reference.

In yet another embodiment, a transgene transferred to present inventioncomprises a gene conferring tolerance to a herbicide and at leastanother nucleotide sequence encoding at least one other trait, such asfor example, an insecticidal protein. Such combination of single genetraits is for example a Cry1Ab gene and a bar gene.

By way of example only, specific events (followed by their APHISpetition numbers) that can be introduced into maize plants include theglyphosate tolerant event GA21 (97-09901p) or the glyphosate tolerantevent NK603 (00-011-01p), the glyphosate tolerant/Lepidopteran insectresistant event MON 802 (96-31701p) Mon810, Lepidopteran insectresistant event DBT418 (96-29101p), male sterile event MS3 (95-22801p),Lepidopteran insect resistant event Bt11 (95-19501p), phosphinothricintolerant event B16 (95-14501p), Lepidopteran insect resistant event MON80100 (95-09301p) and MON 863 (01-137-01p), phosphinothricin tolerantevents T14, T25 (94-35701p), Lepidopteran insect resistant event 176(94-31901p) and Western corn rootworm (04-362-01p), and thephosphinothricin tolerant and Lepidopteran insect resistant eventCBH-351 (92-265-01p).

Maize is used as human food, livestock feed, and as raw material inindustry. Sweet corn kernels having a relative moisture of approximately72% are consumed by humans and may be processed by canning or freezing.The food uses of maize, in addition to human consumption of maizekernels, include both products of dry- and wet-milling industries. Theprincipal products of maize dry milling are grits, meal and flour. Themaize wet-milling industry can provide maize starch, maize syrups, anddextrose for food use. Maize oil is recovered from maize germ, which isa by-product of both dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry. Industrial uses of maize include productionof ethanol, maize starch in the wet-milling industry and maize flour inthe dry-milling industry. The industrial applications of maize starchand flour are based on functional properties, such as viscosity, filmformation, adhesive properties, and ability to suspend particles. Themaize starch and flour have application in the paper and textileindustries. Other industrial uses include applications in adhesives,building materials, foundry binders, laundry starches, explosives,oil-well muds, and other mining applications. Plant parts other than thegrain of maize are also used in industry: for example, stalks and husksare made into paper and wallboard and cobs are used for fuel and to makecharcoal.

The present invention therefore also discloses an agricultural productcomprising a plant of the present invention or derived from a plant ofthe present invention. The present invention also discloses anindustrial product comprising a plant of the present invention orderived from a plant of the present invention. The present inventionfurther discloses methods of producing an agricultural or industrialproduct comprising planting seeds of the present invention, growingplant from such seeds, harvesting the plants and processing them toobtain an agricultural or industrial product.

A deposit of at least 2500 seeds of this invention will be maintained bySyngenta Seed Inc. Access to this deposit will be available during thependency of this application to the Commissioner of Patents andTrademarks and persons determined by the Commissioner to be entitledthereto upon request. All restrictions on availability to the public ofsuch material will be removed upon issuance of a granted patent of thisapplication by depositing at least 2500 seeds of this invention at theAmerican Type Culture Collection (ATCC), at 10801 University Boulevard,Manassas, Va. 20110. The ATCC number of the deposit is PTA-10716. Thedate of deposit was Mar. 10, 2010 and the seed was tested on Mar. 22,2010 and found to be viable. The deposit of at least 2500 seeds will befrom inbred seed taken from the deposit maintained by Syngenta Seed Inc.The ATCC deposit will be maintained in that depository, which is apublic depository, for a period of 30 years, or 5 years after the lastrequest, or for the enforceable life of the patent, whichever is longer,and will be replaced if it becomes nonviable during that period.

Additional public information on plant variety protection may beavailable from the PVP Office, a division of the U.S. Government.

The present invention encompasses a method of producing treated hybridor inbred seed of the present invention and the resultant treated seed.The method includes obtaining seed and treating the seed to improve itsperformance. Hybrid and inbred seed is often treated with one or morepesticides including fungicides, herbicides, herbicidal safeners,fertilizers, insecticides, acaricides, nematocides, bactericides, virusresistant material and other biocontrol agents. Pyrethrins, syntheticpyrethroids, oxadizine derivatives, chloronicotinyls, nitroguanidinederivatives and triazoles, organophosphates, pyrrols, pyrazoles, phenylpyrazoles, diacylhydrazines, biological/fermentation products andcarbamates are used as pesticidal seed treatments. Additionally,Fludioxonil, mefenoxam, azoxystrobin, thiamethoxam, clothianidin and thelike are frequently used to treat maize seed. Methods for treating seedusing fluidized beds, roller mills, rotostatic seed treater and drumcoaster, misting, soaking, filming coating and the like. These methodsof seed treatment are well known in the industry.

The present invention encompasses a hybrid plant with a plant part beingthe segregating grain formed on the ear of the hybrid. This grain is acommodity plant product as are the protein concentrate, protein isolate,starch, meal, flour or oil. A number of different industrial processesextract or utilize these plant products.

Accordingly, the present invention has been described with some degreeof particularity directed to the embodiment of the present invention. Itshould be appreciated, though that the present invention is defined bythe following claims construed in light of the prior art so thatmodifications or changes may be made to the embodiment of the presentinvention without departing from the inventive concepts containedherein.

1. A seed of the maize inbred plant NPAF4467, representative seed ofsaid plant having been deposited under ATCC Accession Number PTA-10716.2. A maize inbred plant NPAF4467, representative seed according to claim1 of said NPAF4467 plant having been deposited under ATCC AccessionNumber PTA-10716.
 3. A plant part of the plant of claim
 2. 4. The plantpart of claim 3, wherein said part is pollen, protoplast, cell, tassel,anther or an ovule.
 5. A maize seed comprising the plant part of claim 3wherein said plant part is a cell.
 6. A process for producing an F1hybrid maize seed, said process comprising crossing a plant of maizeinbred plant NPAF4467 according to claim 2 with a different maize plantand harvesting the resultant F1 hybrid maize seed.
 7. A maize plant orplant part produced by growing the F1 hybrid maize seed of claim
 6. 8.An F1 hybrid maize seed comprising an inbred maize plant cell of inbredmaize plant NPAF4467 according to claim 2, representative seed of saidplant having been deposited under ATCC Accession Number PTA-10716.
 9. Amethod for producing maize seed comprising growing the plant of claim 2until seed is produced, harvesting the seed, wherein the harvested seedis inbred or hybrid or haploid seed.
 10. A seed produced by the methodof claim
 9. 11. A process of introducing a desired trait into maizeinbred plant NPAF4467 comprising: (a) crossing NPAF4467 plants grownfrom NPAF4467 seed, representative seed of which has been depositedunder ATCC Accession Number PTA-10716, with plants of another maizeplant that comprise a desired trait to produce F1 progeny plants,wherein the desired trait is selected from the group consisting of waxystarch, male sterility, herbicide resistance, altered starch, insectresistance, bacterial disease resistance, fungal disease resistance, andviral disease resistance; (b) selecting F1 progeny plants that have thedesired trait to produce selected F1 progeny plants; (c) crossing theselected progeny plants with the NPAF4467 plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait to produce selected backcross progeny plants; and (e)repeating steps (c) and (d) at least three or more times to producebackcross progeny plants that comprise the desired trait and all of thephysiological and morphological characteristics of maize inbred plantNPAF4467 listed in Table 1 when grown in the same environmentalconditions.
 12. A plant produced by the process of claim
 11. 13. A maizeplant having all the physiological and morphological characteristics ofinbred plant NPAF4467, wherein a sample of the seed of inbred plantNPAF4467 was deposited under ATCC Accession Number PTA-10716.
 14. Themaize plant of claim 13, comprising a genome which further comprises atleast one transgene, or a maize plant of claim 13 further exhibiting atrait conferred by a transgene.
 15. The maize plant of claim 14, whereinthe transgene confers a trait selected from the group consisting ofherbicide tolerance; insect tolerance; resistance to bacterial, fungal,nematode or viral disease; waxy starch; altered starch, male sterilityor restoration of male fertility, modified carbohydrate metabolism andmodified fatty acid metabolism.
 16. A method of producing a maize plantderived from the inbred plant NPAF4467, the method comprising the stepsof (a) growing a progeny plant produced by crossing the plant of claim 2with a second maize plant; (b) crossing the progeny plant with itself ora different plant to produce a seed of a progeny plant of a subsequentgeneration; (c) growing a progeny plant of a subsequent generation fromsaid seed and crossing the progeny plant of a subsequent generation withitself or a different plant; and (d) repeating steps (b) and (c) for anadditional 0-5 generations to produce a maize plant derived from theinbred plant NPAF4467.
 17. A method for developing a maize plant in amaize plant breeding program, comprising applying plant breedingtechniques comprising recurrent selection, backcrossing, pedigreebreeding, marker enhanced selection, haploid/dihaploid production, ortransformation to the maize plant of claim 2, or its parts, whereinapplication of said techniques results in development of a maize plant.18. A method of producing a commodity plant product comprising growingthe plant from the seed of claim 10 or a part thereof and producing saidcommodity plant product comprising protein concentrate, protein isolate,starch, meal, flour or oil therefrom.
 19. A method of producing atreated seed of claim 10 comprising obtaining the seed of NPAF4467 andtreating said seed.
 20. A maize seed produced by crossing the plant ofclaim 2 with a different maize plant.